Plant Physiol. (1999) 121: 281-290
Solubilization and Partial Characterization of
Homogalacturonan-Methyltransferase from Microsomal Membranes of
Suspension-Cultured Tobacco Cells1
Florence Goubet2 and
Debra Mohnen*
Complex Carbohydrate Research Center and Department of Biochemistry
and Molecular Biology, University of Georgia, 220 Riverbend Road,
Athens, Georgia 30602-4712
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ABSTRACT |
The
transfer of a methyl group from
S-adenosyl-L-methionine onto the carboxyl
group of 
-1,4-linked-galactosyluronic acid residues in the pectic
polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme
commonly referred to as pectin methyltransferase. A pectin
methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337-347)
and named HGA methyltransferase (HGA-MT). We report the solubilization
of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v)
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
containing 1 mM dithioerythritol. The addition of
phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree
of esterification) to solubilized enzyme increased HGA-MT activity to
35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a
pH optimum of 7.8, an apparent Km for
S-adenosyl-L-methionine of 18 µM, and an apparent Vmax of
0.121 pkat mg
1 of protein. The apparent
Km for HGA and for pectin is 0.1 to 0.2 mg
mL1. Methylated product was solubilized with boiling
water and ammonium oxalate, two conditions used to solubilize pectin
from the cell wall. The release of 75% to 90% of the radioactivity
from the product pellet by mild base treatment showed that the methyl
group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase, and the loss of radioactivity from the product by treatment with pectin methylesterase, demonstrated that the
bulk of the methylated product produced by the solubilized enzyme
was pectin.
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INTRODUCTION |
Pectins are a family of complex polysaccharides present in all
primary walls. To date, three pectic polysaccharides have been identified in all plants investigated: homogalacturonan (HGA), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II) (O'Neill et al., 1990). Each of these pectic polysaccharides can be
O-methylated (O'Neill et al., 1990; An et al., 1994
) and
O-acetylated (Ishii, 1997). For example, the -1,4-linked
D-galactosyluronic acid residues of HGA (O'Neill
et al., 1990) and RG-II (Pellerin et al., 1996) are partly
methylesterified, whereas RG-I contains 4-O-methyl-GlcUA (An
et al., 1994) and RG-II contains 2-O-methyl-Fuc and
2-O-methyl-Xyl (O'Neill et al., 1990, 1996).
Enzymes that methylate pectin, typically referred to as pectin
methyltransferases (PMTs), have been studied in mung bean (Kauss et
al., 1967; Crombie and Reid, 1998), flax (Vannier et al., 1992), tomato
(Crombie and Reid, 1998), and tobacco (Nicotiana tabacum) (Goubet et al., 1998). These PMTs use
S-adenosyl-L-methionine (SAM) as a
methyl donor in vitro. The main difference reported between these PMTs
is the effect of the addition of exogenous methyl acceptors, pectin or
HGA, on in vitro membrane-bound PMT activity. The membrane-bound PMT
from mung bean is activated by HGA (Kauss et al., 1967), whereas the
membrane-bound PMT from flax is activated by HGA following freezing and
thawing of the membranes (Bruyant-Vannier et al., 1996). The
membrane-bound PMT from tobacco is not activated by HGA or pectin,
irrespective of whether the membranes have been frozen or not (Goubet
et al., 1998).
The PMT from flax has been solubilized with 0.5% (w/v) Triton X-100,
and the solubilized enzyme is activated by HGA (Bruyant-Vannier et al.,
1996). The amount of activation by HGA of solubilized flax PMT and
previously frozen membrane-bound flax PMT are comparable (Bruyant-Vannier et al., 1996; Bourlard et al., 1997). Bruyant-Vannier et al. (1996) reported that flax PMT had a pH optimum of 7.1, an
apparent Km for SAM of 0.5 µM, and an apparent
Km for HGA of 0.5 to 0.7 mg
mL1. However, Bourlard et al. (1997) more
recently reported that the solubilized- and HGA-activated PMT from flax
has a pH optimum of 5.5, an apparent
Km for SAM of 20 µM, and a Km
for HGA was 0.25 mg mL1. The reasons for the
differences in the pH optimum and kinetic constants for PMT(s) from
flax have not been explained.
In vitro studies of HGA biosynthesis suggest that the galactosyluronic
acid residues in HGA are methylesterified after polymerization of at
least some of the GalA residues in the HGA chain (Kauss and Swanson,
1969; Doong et al., 1995; Goubet et al., 1998). The glycosyltransferase
that synthesizes HGA, HGA 4--galacturonosyltransferase (HGA-GalAT),
has been studied in mung bean (Villemez et al., 1966), sycamore
(Bolwell et al., 1985), and tobacco (Doong et al., 1995). The tobacco
enzyme has been characterized in microsomal membranes (Doong et al.,
1995), and a galacturonosyltransferase has been solubilized from these
membranes using the detergent CHAPS (Doong and Mohnen, 1998). HGA with
degrees of polymerization (DP) greater than 9 are required as exogenous
acceptors to obtain appreciable solubilized galacturonosyltransferase
activity. Membrane-bound HGA-GalAT generates a product that is
approximately 50% esterified (Doong et al., 1995); at least 40% of
the esters have been shown to be methyl esters (Doong et al., 1995).
The notion that HGA is methylesterified following its polymerization is
supported by the finding that the in vitro methylation of HGA in
microsomes by SAM is increased by the inclusion of UDP-GalA (Goubet et
al., 1998). In contrast, the methyl donor SAM does not increase the rate of HGA formation by HGA-GalAT (Kauss and Swanson, 1969; Doong et
al., 1995).
We recently identified a PMT in microsomal membranes of
suspension-cultured tobacco cells (Goubet et al., 1998). The PMT
methylates HGA and has been named HGA methyltransferase (HGA-MT) to
distinguish it from methyltransferases that methylate the pectic
polysaccharides RG-I and RG-II. We now describe the solubilization of
tobacco HGA-MT and the partial characterization of the solubilized
enzyme and the products its synthesizes.
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MATERIALS AND METHODS |
Chemicals
The chloride salt of
S-adenosyl-L-Met, GalA, tri-GalA, the
ammonium salt of UDP-GalA, polygalacturonic acid (referred to as HGA in
this paper), DL-phosphatidylcholine,
DL-phosphatidylinositol, and
DL-phosphatidylethanolamine were purchased
from Sigma. Pectins of 31% to 93% degrees of esterification (DE) were
purchased from Sigma or obtained as a gift from the Hercules
Corporation (Wilmington, DE). Dextran standards were purchased from
Pharmacia.
[14C]Methyl-S-adenosyl-L-Met
(specific activity 55 mCi/mM) was purchased from
Amersham. CHAPS was purchased from Pierce. Triton X-100, Triton X-114,
and Genapol X-080 were purchased from Boehringer Mannheim.
Oligogalacturonides of DP 7 to 23 were prepared as described (Doong et al., 1995). Partially purified decagalacturonides and pentagalacturonides were prepared by fractionation of
oligogalacturonides (DP 7-23) using Q-Sepharose anion-exchange resin
as described previously (Lo et al., 1994; Doong and Mohnen, 1998).
Plant Material
Suspension-cultured tobacco (Nicotiana tabacum L. cv
Samsun) cells, originally isolated from pith callus tissue (Eberhard et
al., 1989), were grown on Murashige and Skoog medium supplemented with
4.5 µM 2,4-D and 90 mM
Suc and subcultured every 13 to 14 d (Doong et al., 1995).
Preparation of Membranes from Tobacco Cell Suspension Cultures
Membranes were prepared by a modification of the method of
Villemez et al. (1966). Tobacco cells (75 g) were collected by filtration 3 or 4 d after transfer to fresh medium. The cells were
homogenized with a polytron at maximum speed for 2 min at 4°C in 100 mL of grinding buffer (50 mM Tris-HCl, pH 7.3, 0.4 M Suc, 1% (w/v) BSA, and 1 mM EDTA). The
homogenate was strained through a nylon cloth (50-µm pore size) and
the filtrate centrifuged at 3,500g for 15 min. The
supernatant was centrifuged for 1 h at 100,000g to
produce a membrane pellet. The pellet was resuspended with a
Potter-Elvehjem glass homogenizer (Fisher Scientific) in 5 mL of
storage buffer (0.4 M Suc and 50 mM Tris-HCl, pH 6.8) and kept at 
80°C prior
to use. The protein content of the membranes was determined using a
Bradford (1976) assay with BSA as a standard.
Solubilization of Membrane Proteins
Membranes (1 mL, 4 mg mL1 of protein) were
mixed with 1 mL of 1.3% (w/v) CHAPS and 1 mM
dithioerythritol (DTE), vortexed, and the suspension incubated for 2 min at 4°C. The suspension was then centrifuged for 45 min at
180,000g. The supernatant was used as solubilized enzyme.
The pellet was resupended in 1 mL of storage buffer (0.4 M Suc and 50 mM Tris-HCl,
pH 6.8) and used as non-solubilized enzyme. The protein content of the
soluble fraction and the pellet were determined using the method of
Bradford (1976) and DC protein assays (Bio-Rad) with BSA as a standard.
The relative protein content of the soluble versus membrane proteins
were the same using these two assays. The Bradford assay gave absolute
protein values 33% lower that the DC assay.
HGA-MT Assay
The HGA-MT assay was a modification of that described by Kauss and
Hassid (1967). An aliquot (25 µL, approximately 25 µg of protein)
of the soluble fraction or resuspended membrane pellet was added to 25 µL of concentrated reaction buffer (50 mM Tris-MES, pH
8.5, 25 µg DL-phosphatidylcholine, 0.4 M Suc,
50 µg HGA or pectin of 30% DE, 8 µM
[14C]methyl-SAM [0.01 µCi], and 12 µM SAM) and incubated at 30°C for 4 h. The
reaction was stopped and the methylated products precipitated by the
addition of 50 µL of 20% (w/v) TCA and 5 µL of a 10% (w/v) BSA
solution. The resulting suspensions were centrifuged 5 min at
4,000g. Unincorporated SAM was removed by washing the pellets twice with 200 µL of 2% (w/v) TCA. The washed pellets were
resuspended in 300 µL of water, and the radioactivity incorporated into product was measured by liquid scintillation counting using scintillation cocktail (Scintiverse BD, Fisher Scientific).
Uronic Acid Assay
Uronic acids were determined by a
meta-hydroxybiphenyl assay (Blumenkrantz and
Asboe-Hansen, 1973).
Chemical Extraction of Radiolabeled Product
The pellets obtained after TCA precipitation were treated with
boiling water, 0.5% (w/v) boiling ammonium oxalate, or 0.1 N NaOH at 20°C to 25. The water and oxalate treatments
were performed three times for 1 h. The NaOH treatment was
performed for 4 h, followed by neutralization with 0.1 N HCl. The suspensions were centrifuged, and the amount of
radioactivity in the supernatant and pellet determined.
EPGase and Pectin Methylesterase (PME) Digestion of
Product
Methylated product was demethylesterified by treatment for 4 to
12 h at 25°C with 1 to 4 units (µmoles of methanol released per minute) of a purified cloned Aspergillus aculeatus PME
expressed in Aspergillus oryzae (Christgau et al., 1996).
Methylated product was fragmented by treatment for 4 to 12 h at
25°C with 1 to 4 units (micromoles of reducing ends produced per
minute) of a purified EPGase from Aspergillus niger. Both
enzymes were purified from culture filtrates (gifts of Carl Bergmann,
Complex Carbohydrate Research Center, Athens, GA).
HPLC of 14C-Labeled Products
Radiolabeled methanol released from the
14C-labeled product by treatment with PME or NaOH
was collected by distillation and separated using a Rezex ROA-organic
acid column (Phenomenex, Torrance, CA) eluted with water (flow rate 0.6 mL min1) on a chromatography system (DX 500, Dionex). Non-radiolabeled methanol was detected by electrochemical
detection with post-column addition of 400 mM NaOH (0.2 mL
min1). Fractions were collected manually and
radioactivity was determined by liquid scintillation counting using
scintillation cocktail (Fisher Scientific).
Size-Exclusion Chromatography of 14C-Labeled
Products
Radioactive products obtained after TCA precipitation in
the HGA-MT assay were solubilized with 0.3 M potassium
acetate, pH 5.2, containing 6 mM imidazole. Half of the
solubilized product was treated for 8 h with 2 units of a purified
EPGase (748 units mL1) from Fusarium
moniliforme (gift of Carl Bergmann, Complex Carbohydrate Research
Center, Athens, GA). Intact and digested product, non-radiolabeled pectin, and HGA standards were filtered through 0.2-µm nylon
membranes (Rainen Instrument, Woburn, MA) and then fractionated using a Superose 12HR 10/30 size-exclusion column (Pharmacia) in 0.3 M potassium acetate, pH 5.2 (flow rate 0.48 mL
min1). Fractions (0.48 mL) were collected and
radioactivity measured by liquid scintillation counting using
scintillation cocktail (Fisher Scientific). The uronic acid content of
non-radiolabeled standards separated under the same conditions was
determined colorimetrically (Blumenkrantz and Asboe-Hansen, 1973).
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RESULTS |
Solubilization of HGA-MT Activity and Optimization of the
Solubilization Procedure
Solubilization of HGA-MT Using CHAPS
Four detergents previously shown to be useful for the
solubilization of membrane-bound enzymes (Jones et al., 1987; Waldron et al., 1989), Triton X-100, Triton X-114, CHAPS, and Genapol X-080,
were separately added to tobacco membranes at a final concentration of
0.5% (2 mg mL1 of protein). CHAPS, Triton
X-100, and Triton X-114 solubilized an average of 6% of the total
HGA-MT activity. No solubilized HGA-MT activity was recovered using
Genapol X-080. The solubilization of HGA-MT by CHAPS was optimized
since it caused the least loss of total HGA-MT activity in the
detergent-dispersed enzyme and because the high critical micelle
concentration (CMC) of CHAPS would facilitate the effective removal of
the detergent by dialysis during protein purification.
The maximal solubilization of HGA-MT was obtained with 0.6% to 0.7%
(w/v) CHAPS (Fig. 1), corresponding to
1.2 to 1.5 times CMC. The solubilized HGA-MT activity was inhibited in
a concentration dependent manner at greater than 1.2 to 1.5 times the
CMC of CHAPS. Thus, 0.65% (final concentration, w/v) CHAPS was used to
solubilize HGA-MT.
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| Figure 1.
Effect of CHAPS concentration on the
solubilization of HGA-MT. The data are the average counts per minute
(±SE) of product recovered from duplicate samples from at
least four independent experiments except for the 0.1% and 1% (w/v)
CHAPS points, which are from duplicate samples from one
experiment.
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The highest recovery of HGA-MT activity was obtained with 2 min of
CHAPS treatment. When the solubilization time was increased the amount
of solubilized HGA-MT activity decreased (Fig.
2). The addition of detergent followed by
the immediate centrifugation of the membranes resulted in a low
recovery of HGA-MT. The HGA-MT activity after a 2-min solubilization
time was stable for at least 5 d at 3°C, whereas the HGA-MT
after a 30-min solubilization was unstable, with only 50% of the
HGA-MT activity recovered after 2 d at 3°C.
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| Figure 2.
Effect of solubilization time on the
solubilization of HGA-MT. The data are the average counts per minute
(±SE) incorporated into product in triplicate samples from
one experiment for the 0-, 30-, and 60-min points and from three
independent experiments for the other points.
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Optimization of the HGA-MT Solubilization Protocol
The inclusion of the reducing agent DTE during enzyme
solubilization increased the amount of total solubilized HGA-MT
activity recovered to 159% ± 25%, (mean ± SD, 0.5 mM DTE) and 161% ± 24% (5 mM DTE) of that
recovered upon solubilization in the absence of DTE. Low concentrations
of the Cys and Ser protease inhibitor PMSF (0.1 mM) did not
affect the solubilization of HGA-MT activity. In contrast, inclusion of
1 mM PMSF and 5 mM EDTA in the solubilization buffer reduced the amount of total HGA-MT activity recovered to 83% ± 12% and 87% ± 3%, respectively. Low concentrations of
MnCl2 (0.05-0.5 mM) during
solubilization did not affect the recovery of solubilized HGA-MT, but
the recovered enzyme activity was lowered to 50% at 5 mM
and 30% at 10 mM MnCl2.
MgCl2 (12 mM) reduced the amount of
total solubilized HGA-MT activity by 30%. The amount of total HGA-MT
activity recovered was not increased by the addition of 0.1 mg
mL1 of HGA or 2.5 to 10 µM
UDP-GalA in the solubilization buffer (data not shown).
Under our experimental conditions, the most effective procedure for the
solubilization of HGA-MT from tobacco membranes was to treat the
membranes (4 mg mL1 of protein) with 1 mL of
1.3% (w/v) CHAPS and 1 mM DTE for 2 min at 4°C, followed
by centrifugation for 45 min at 180,000g.
Optimization of the HGA-MT Assay for Solubilized HGA-MT
Precipitation of the Methylated Product Generated by
Solubilized HGA-MT
A protein carrier (e.g. BSA) and a short precipitation period
increased the yield of product generated by solubilized HGA-MT (Table
I). Attempts to precipitate the product
produced by solubilized HGA-MT using ethanol (Vannier et al., 1992) and
methanol/chloroform (Doong et al., 1995) were not effective.
Methanol/chloroform precipitation followed by ethanol washes gave
variable counts per minute with high background counts per minute, and
the precipitation of the product by ethanol followed by ethanol-NaCl
washes gave a low yield of product (results not shown). The method used
to assay solubilized HGA-MT was the precipitation of product with 20%
(w/v) TCA containing 500 µg of BSA and the removal of unincorporated SAM by washing the pellet twice with 2% (w/v) TCA.
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Table I.
Optimization of the precipitation procedure for
recovery of the products generated by HGA-MT
Solubilization was performed with 0.65% (w/v) CHAPS for 2 min at
4°C. After centrifugation, the solubilized HGA-MT in the supernatant
was incubated for 4 h in reaction buffer containing 10 µM SAM (20,000 cpm [14C]SAM), 50 µg HGA,
or pectin (30% DE), and 25 µg of phosphatidylcholine, and the
methylated products were precipitated by 20% (w/v) TCA with or without
BSA for 5 min or 12 h. The solutions were centrifuged and the
pellets were washed twice with 200 µL of 2% (w/v) TCA. The data are
the averages ± SD from triplicate samples from at
least two independent experiments. n.d., Not determined.
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Effect of Potential Stimulatory Agents on Solubilized HGA-MT
Activity
To test the effect of the reducing agent DTE on HGA-MT activity,
HGA-MT activity in reactions with and without 1 mM DTE was assayed using HGA-MT solubilized in the absence of DTE. The inclusion of DTE in the reaction did not inhibit or stimulate solubilized HGA-MT
activity. Thus, the stimulatory effect achieved by adding DTE during
the solubilization of HGA-MT was likely due to a stabilization of the
solubilized enzyme and not to an activation of the enzyme.
MgCl2 (1 mM) inhibited the
solubilized HGA-MT by 60%, while Mn activated the solubilized HGA-MT
with maximum activation (1.7-fold) at 10 to 20 mM
MnCl2. MnCl2 inhibits
HGA-MT at concentrations above 50 mM. Solubilized HGA-MT
was not affected by GTP or ATP at 0.1 to 1 mM, and was
inhibited by 50% at 10 mM.
The ability of exogenous lipids to stimulate solubilized HGA-MT
activity was determined. Phosphatidylcholine and
phosphatidylethanolamine (0.25-0.5 mg mL1)
activated HGA-MT activity by 20% to 45%, and 10% to 15%,
respectively. Phosphatidylinositol (0.5 mg mL1)
inhibited solubilized HGA-MT by 50%. At concentrations greater than
0.5 mg mL1 all the lipids inhibited HGA-MT
activity.
Effect of Potential Substrates on Solubilized HGA-MT Activity
The effect of the exogenous acceptors HGA and pectin on total
solubilized HGA-MT activity was determined. HGA (DE = 0) and pectin with 30% DE both increased total solubilized HGA-MT activity (Table II). The ability of pectins with
higher DE to increase total solubilized HGA-MT activity could not be
determined, since TCA does not effectively precipitate pectins with a
high DE (10% of pectin [55%-73% DE] precipitated; 30% of pectin
[93% DE] precipitated) or a low
Mr (20% of OGAs [DP 7-23]
precipitated).
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Table II.
Effect of pectins with different degrees of
methylesterification and oligomerization on total solubilized HGA-MT
activity and a comparison of their precipitation by TCA
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Attempts to increase HGA-MT activity further by using pectin in
combination with MnCl2 resulted in a 70% to
100% decrease in total HGA-MT compared with the activity detected in
absence of these compounds. In fact, Mn made the pectin gel at
concentrations of 0.1 to 50 mM MnCl2
and 0.1% to 1% (w/v) HGA or pectin (30% DE). UDP-GalA, an activator
of membrane-bound HGA-MT (Goubet et al., 1998), did not stimulate
solubilized HGA-MT activity either in the presence or absence of
MnCl2.
In summary, approximately half of the protein and 30% to 40% of the
total HGA-MT activity was solubilized using the HGA-MT solubilization
and assay procedures summarized in Table
III.
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Table III.
Recovery of total HGA-MT activity and
fractionation of protein during the solubilization of HGA-MT
The percentage of solubilized and membrane-bound (pellet) HGA-MT
activities in the presence of HGA were compared with the membrane-bound
HGA-MT activity. The data are the averages ± SE of
protein and HGA-MT activity values from triplicate samples from at
least six independent experiments.
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Characterization of Solubilized HGA-MT
The temperature optimum, pH optimum, and enzyme kinetics were
established for solubilized HGA-MT in the presence of exogenous HGA and
independently for solubilized HGA-MT in the presence of exogenous
pectin (30% DE). The characteristics of solubilized HGA-MT acting on
the two different exogenous acceptors were similar. The combined
results from reactions containing either HGA or pectin (30% DE) as an
exogenous acceptor are presented.
The temperature optimum for solubilized HGA-MT was between 25°C and
30°C. Solubilized HGA-MT was completely inactivated by treatment for
1 h at 60°C or for 5 min at 100°C. Solubilized HGA-MT was
stable for at least 1 month at 80°C, and for up to 5 d when stored at 3°C, but was reduced by 50% when stored at 6°C for
1 d. Similar levels of stability for frozen xyloglucan
fucosyltransferase (Hanna et al., 1991) and glucuronosyltransferase
(Waldron et al., 1989) have been reported.
The effect of reaction pH ranging from 5.5 to 8.6 on solubilized HGA-MT
activity was determined (Fig. 3). A major
peak of activity was obtained at pH 7.8 to 8.0, and minor peaks
occurred at pH 6.8 to 7.3 and pH 8.0 to 8.3.
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| Figure 3.
Effect of pH on solubilized HGA-MT activity. The
data are the average counts per minute (±SE) incorporated
into product in duplicate samples from five independent experiments.
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The rate of incorporation of 14C into product was
linear during the first 4 h (Fig.
4). The apparent
Km of solubilized tobacco HGA-MT for
SAM was 18 ± 3 µM (Fig.
5A). The apparent
Km of solubilized HGA-MT for HGA or
pectin (30% DE) was 0.1 to 0.2 mg mL1 (Fig.
5B), which represents approximately 21 µM HGA
assuming an average DP of 40 for HGA. The apparent
Vmax of solubilized HGA-MT was
0.121 ± 0.02 pkat mg1 of protein (Fig.
5).
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| Figure 4.
Time course of the incorporation of
14C-methyl into precipitable product. The data are the
average counts per minute (±SE) incorporated into product
in duplicate samples from three independent experiments.
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| Figure 5.
Hanes-Woolf plot of the production of methylated
product by solubilized HGA-MT. A, Hanes-Woolf plot for SAM. Reactions
contained with 1 mg mL1 of pectin (HGA or pectin [30%
DE]). B, Hanes-Woolf plot for pectin (HGA and pectin [30% DE]).
Reactions contained 100 µM SAM. The data represent the
average initial velocities (±SE) in duplicate samples from
at least two independent experiments. Initial velocity (V0)
is in picomoles of methyl incorporated per second per milligram of
protein.
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Characterization of the Total Product Produced by Solubilized
HGA-MT
The products synthesized by solubilized enzyme in the absence of
exogenous acceptor (i.e. only endogenous acceptor present) or in the
presence of exogenous HGA and pectin (30% DE) were partially solubilized by boiling water and boiling ammonium oxalate (Table IV). These same two treatments partially
solubilized the products generated by membrane-bound HGA-MT (Goubet et
al., 1998) and are commonly used to solubilize pectin from isolated
cell walls (O'Neill et al., 1990; Goubet et al., 1993). Water
solubilized a similar percentage of the product (77%-81%) produced
using endogenous acceptor or exogenous HGA, while only 55% of the
product produced using pectin (30% DE) was solubilized (Table IV).
Ammonium oxalate solubilized a greater percent of the product
synthesized using exogenous HGA (72%) compared with product made using
endogenous acceptor (40%) or pectin of 30% DE (53%). Between 75%
and 90% of the radioactivity in the total product produced using HGA, pectin (30% DE), or endogenous acceptor (Table IV) was released by
treatment with 0.1 N NaOH. The product was identified as
methanol by HPLC (data not shown). Thus, the bulk (>75%) of the
methyl group was incorporated as a methyl ester rather than as a methyl ether.
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Table IV.
14C-Methylated product solubilized
using different chemical treatments
Results are the percentages of radioactivity solubilized from the
product pellet compared with the radioactivity present in the pellet
before treatment. Results from reactions with endogenous substrate and
HGA as exogenous substrate are the averages ± SD of
the percentage of cpm of product solubilized from at least two samples
from at least two independent experiments. Results for reactions with
pectin of 30% DE as exogenous substrate are the average percentage cpm
of product solubilized from duplicate samples from one experiment.
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Additional evidence that the radiolabeled product was indeed
methylesterified HGA was obtained by treating
14C-methylated product with EPGase and PME. HGA
and pectin (30% DE) are precipitated by TCA (Table II) while OGAs and
highly methylated pectin are not effectively precipitated. Thus,
radiolabeled and partially methylated HGA oligomers released from HGA
by EPGase or methanol released from the product by PME would not be
precipitated by TCA. The material generated by the solubilized enzyme
was resuspended in water or treated with EPGase or PME and then
precipitated by with TCA. Approximately 37% of the product produced
using endogenous acceptor or exogenous HGA acceptor was not
precipitated by TCA, indicating that it had been fragmented by EPGase
(Table V). These results suggest that at
least 38% of the product produced in the presence of exogenous HGA was
methylated pectin. Further evidence that
14C-methyl was incorporated into HGA was
obtained by the treatment of the 14C-methylated
product with PME. Approximately 33% to 37% of the radioactivity was
not precipitated following PME treatment (Table V). Since the PME used
here catalyzes the hydrolysis of 75% to 85% of the methyl groups in
highly esterified pectin (Christgau et al., 1996), these results
confirm that at least 37% of the product produced in the presence of
exogenous HGA is methylated pectin.
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Table V.
Fragmentation of intact methylated product by EPGase
and PME
The cpm ± SE recovered upon precipitation of intact
or enzyme-treated product is shown. Results are the average ± SD cpm recovered from at least duplicate samples from at
least two independent experiments.
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Characterization of the Water- and Oxalate-Solubilized Product
Produced by Solubilized HGA-MT
The ability of EPGase and PME to fragment
14C-labeled product solubilized from the total
product pellet by boiling water or boiling oxalate (see Table IV) was
also tested (Table VI). The bulk (>70%)
of the EPGase-treated water- and oxalate-solubilized product produced
using endogenous acceptor or exogenous HGA as an acceptor was not
precipitated by TCA. The susceptibility to EPGase suggests that 55% to
70% of the total product produced by solubilized HGA-MT in the
presence of endogenous acceptor or exogenous HGA was pectin.
Sensitivity of the water- and oxalate-solubilized product to PME
treatment confirms that the bulk (>64%) of the solubilized product
was pectin and that at least 51% of the total product was sensitive to
PME. The fact that a greater percentage of the water- and
oxalate-solubilized product compared with total product was sensitive
to hydrolysis by EPGase and PME likely reflects a greater accessibility
of the enzymes to products that have been extracted from total product
prior to enzyme treatment. Interestingly, a smaller percentage of the
product methylated in the presence of exogenous pectin (30% DE) was
not precipitated following treatment of water- and oxalate-solubilized
product with EPGase (53%) and PME (43%). These results suggest that
either the exogenous pectin (30% DE) itself is not as amenable as HGA
or the endogenous acceptor to complete hydrolysis by these enzymes, or
that the in vitro-methylated pectin is not as accessible.
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Table VI.
Fragmentation of oxalate- and water-solubilized
methylated product by EPGase and PME
The cpm ± SD recovered upon precipitation of intact
or enzyme-treated product is shown. Results are the average ± SD cpm from at least two samples from at least two
independent experiments. Results for reactions with pectin (30% DE) as
exogenous acceptor are the average cpm from duplicate samples from one
experiment.
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The intact and EPGase-treated radiolabeled products produced by the
solubilized enzyme in the presence of exogenous pectin (30% DE)
acceptor were separated by size-exclusion chromatography over a
Superose HR 10/30 column. The retention times of the radiolabeled product were compared with the retention times of commercially available pectin (30% DE) and HGA (data not shown). We were unable to
recover intact radiolabeled methylated product from the size-exclusion column despite repeated attempts using different chromatographic conditions. At least half of the intact radiolabeled product bound to
the spin filter used to remove particulates prior to injection onto the
column. The remaining radioactive product apparently remained bound to
the column. However, EPGase treatment of the intact product gave peaks
that co-eluted with the intact pectin acceptor, others that co-eluted
in the range of oligogalacturonides of DP 2 to >15, and some
radioactivity that eluted with retention times similar to Glc and
methanol. For example, in one reaction with pectin (30% DE) as the
exogenous acceptor, 63% of the EPGase-fragmented product recovered
from the column eluted as radioactive peaks with retention times
between those of the exogenous pectin (30% DE) acceptor and
digalacturonic acid. The appearance of radioactive peaks following
EPGase treatment of intact 14C-labeled
product was reproducible, although the retention times of the
radioactive peaks and the amount of radioactivity recovered varied
between different experiments. Nevertheless, these results confirm that
solubilized HGA-MT methylates pectin and suggest that the degree and/or
pattern of HGA methoxylation may vary in product produced in different
in vitro enzymatic reactions.
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DISCUSSION |
HGA-MT transfers a methyl group from SAM onto the carboxyl group
of GalA in the HGA of pectin (Goubet et al., 1998). Solubilization of
the membrane-bound HGA-MT from tobacco with the detergent CHAPS resulted in a reduction of total HGA-MT activity. Detergents also inhibit membrane-bound PMT in flax (Bruyant-Vannier et al., 1996). The
activity of solubilized tobacco HGA-MT is increased by the addition of
HGA or pectin (30% DE), which is consistent with the identification of
the solubilized enzyme as HGA-MT. The described solubilization and
assay procedures allow for the recovery of approximately 35% of the
tobacco membrane-bound HGA-MT activity as soluble enzyme.
The concentration of detergent is a critical condition for the
solubilization of membrane-bound enzymes (Wasserman et al., 1989; Doong
and Mohnen, 1998). The solubilization of HGA-MT was optimal at 0.65%
(w/v) CHAPS, a concentration corresponding to 1.2 times the CMC. A
similar effective CHAPS concentration was reported for the
solubilization of glucan synthase, which was optimal at 0.6% (w/v)
CHAPS (Wasserman et al., 1989). Bruyant-Vannier et al. (1996) reported
the solubilization of flax PMT using 1% (w/v, 100 times the CMC)
Triton X-100, however, no optimization of the concentration of Triton
X-100 was presented.
Reducing agents, protease inhibitors, and chelators have been reported
to increase the solubilization and/or the stability of membrane-bound
enzymes (Wu et al., 1991; Von Jagow and Schägger, 1994). The
inclusion of 0.5 to 5 mM DTE during solubilization increased the amount of solubilized HGA-MT activity recovered by 60%.
In contrast, neither PMSF or EDTA increased the total solubilized
HGA-MT activity. The activity of some soluble enzymes is increased by
the presence of divalent cations during solubilization (Doong and
Mohnen, 1998). MgCl2 and
MnCl2 had no affect on membrane-bound HGA-MT
(Goubet et al., 1998). On the contrary, solubilized HGA-MT activity was
inhibited by 1 mM MgCl2 and activated
by 10 to 20 mM MnCl2. Some enzymes
require nucleotides as cofactors (Fevre, 1983; Girard and Fevre, 1991).
However, HGA-MT was not stimulated by GTP or ATP.
The methylation of HGA is believed to occur after the HGA chain is
polymerized (Kauss and Swanson, 1969; Doong et al., 1995; Goubet et
al., 1998). Thus, it was possible that the inclusion of the substrates
for HGA synthesis, HGA and UDP-GalA, in the solubilization buffer would
stabilize HGA-MT. However, the inclusion of UDP-GalA or HGA during
solubilization had no effect on the amount of HGA-MT activity
recovered.
Phospholipids are often required to stabilize solubilized
membrane-bound enzymes. The stabilization can require one phospholipid or a mixture of phospholipids (Wasserman and McCarthy, 1986; Jones et
al., 1987; Kasamo and Nouchi, 1987; Kasamo and Sakakibara, 1995).
Solubilized tobacco HGA-MT activity was increased by the addition of
phosphatidylcholine or phosphatidylethanolamine, whereas phosphatidylinositol inhibited HGA-MT activity. Positively charged phospholipids also enhance the recovery of solubilized glucan synthase
activity from red beet (Wasserman and McCarthy, 1986) and soybean
(Kauss and Jeblick, 1986).
We previously showed that TCA effectively precipitates the products
generated by HGA-MT in tobacco cell membranes (Goubet et al., 1998),
and that alternative procedures to precipitate pectin using ethanol
(Vannier et al., 1992) and methanol/chloroform (Doong et al., 1995)
were not effective. The latter two procedures were also not effective
for the reproducible recovery of methylated product generated by
solubilized HGA-MT. However, appreciable methylated product produced by
solubilized HGA-MT was recovered by precipitation with TCA in the
presence of BSA. No carrier was required to facilitate the
precipitation of product synthesized by membrane-bound HGA-MT, and the
length of the precipitation time was not critical. In contrast, the
inclusion of BSA during TCA precipitation and a short precipitation
time resulted in a greater, less variable recovery of product produced
by solubilized HGA-MT. The loss of methyl groups by de-esterification
in acid conditions (BeMiller, 1986) could account for the loss of
radioactivity upon extended exposure of the product to acidic
solutions. The methylated products produced by solubilized HGA-MT were
in direct contact with the TCA, while the products produced by
membrane-bound HGA-MT were likely surrounded by a membrane. Also, the
protein content of the solubilized HGA-MT fraction (1 mg
mL1 of protein) was lower than that in
membranes (4 mg mL1 protein) and may lead to a
less-effective coprecipitation of product by TCA.
Microsomal membranes contain endogenous polysaccharides (Goubet and
Morvan, 1993). The lack of effect of exogenous pectin on membrane-bound
HGA-MT may be due to the presence of endogenous polysaccharides in the
membranes (Goubet et al., 1998) or to the inaccessibility of the
exogenous pectin to the HGA-MT in the membranes. Upon solubilization of
HGA-MT from the membranes, the endogenous methyl acceptors may
dissociate from the membranes and become less accessible to HGA-MT. The
results presented here suggest that the solubilization of membranes
results in the dilution of exogenous acceptor and a concomitant
reduction in total HGA-MT activity. The total HGA-MT activity was
increased by the addition of HGA or pectin (30% DE), suggesting that
these polysaccharides act as exogenous acceptors for the enzyme. The DE
of pectin has been proposed to be an important factor in pectin
methylation (Bourlard et al., 1997). Solubilized tobacco HGA-MT
activity was greatest when HGA or pectin with a low DE were used as
exogenous acceptors. However, since the TCA procedure used to recover
methylated product does not completely precipitate pectin with high
DEs, we could not address whether pectin of high DE serves as an
acceptor for HGA-MT.
UDP-GalA is an activator of membrane-bound HGA-MT (Goubet et al., 1998)
that is thought to act by increasing the amount of HGA synthesized by
HGA-GalAT (Doong and Mohnen, 1998), thereby increasing the amount of
HGA available to membrane-bound HGA-MT. UDP-GalA did not stimulate
solubilized HGA-MT activity either in the presence or absence of
MnCl2, a cation required to detect HGA-GalAT
activity (Doong et al., 1995; Doong and Mohnen, 1998).
The characteristics of the solubilized HGA-MT from tobacco are similar
to membrane-bound HGA-MT (Table VII). The
temperature and pH optimum for these enzymes are 25°C to 30°C and
7.8 to 8.0, respectively. A minor peak of activity for solubilized
HGA-MT was observed at pH 7.3. A similar peak was observed with the
membrane-bound HGA-MT (Goubet et al., 1998). The apparent
Km of solubilized HGA-MT for SAM (18 µM) was comparable to the apparent
Km (38 µM) for the membrane-bound HGA-MT (Goubet et al., 1998). Determination of the
Km of membrane-bound HGA-MT for pectin
was not successful, since the membrane-bound enzyme was not dependent
upon exogenous pectin (Goubet et al., 1998). The apparent
Km of solubilized HGA-MT was similar
for HGA and pectin (30% DE) (0.1-0.2 mg mL1).
The apparent Vmax of 0.121 pkat
mg1 of protein for solubilized HGA-MT was
7-fold lower than the apparent Vmax of
0.81 pkat mg1 of protein for the membrane-bound
enzyme (Goubet et al., 1998).
We do not know the reason for the reduction in HGA-MT activity upon
solubilization from the membrane. Partial denaturation of the enzyme by
the detergent and a dilution or loss of the endogenous acceptor could
be lead to reduced activity. HGA and pectin (30% DE) may not be
optimal substrates for the enzyme. Another possibility is that the
methylation of pectin in planta may occur in an enzyme complex
containing HGA-MT and HGA-GalAT, the galacturonosyltransferase that
synthesizes HGA (Doong et al., 1995). The existence of such a complex
is supported by the stimulation of membrane-bound HGA-MT by UDP-GalA,
the substrate for HGA-GalAT. The lack of stimulation of solubilized
HGA-MT by UDP-GalA may indicate a disassembly of a HGA-MT/HGA-GalAT
complex upon solubilization of the enzymes from the membrane. Such a
disruption of a complex could lead to a concomitant reduction in the
accessibility of HGA-MT to the HGA/pectin substrate with a resulting
reduction in the apparent Vmax.
The hydrolysis of 75% to 90% of the radioactive product produced by
the solubilized enzyme by mild base treatment, and the resulting
formation of methanol, indicates that 
75% of the methyl group was
incorporated as a methoxyl group, as expected for methylesterified HGA.
The remaining 10% to 25% of the radiolabeled product may represent
methyl group incorporated as a methyl ether into other compounds such
as RG-I (An et al., 1994), RG-II (O'Neill et al., 1990, 1996), or
plant metabolites (Chiang et al., 1996).
The bulk (53%) of radiolabeled products produced by the solubilized
enzyme were shown to be methylated pectin, based on their sensitivity
to PME and EPGase. Treatment of 14C-methylated
product with EPGase generated radiolabeled products of a size range
expected for pectic fragments. At present we cannot exclude the
possibility that some small percentage of the methoxylated product
produced by the solubilized enzyme may represent methoxylation of a
substrate other than pectin. For example, a prenylcysteine carboxyl
methyltransferase (Hrycyna and Clarke, 1990) that methoxylates farnesylcysteine and geranylgeranylcysteine has recently been characterized in tobacco (Crowell et al., 1998).
The solubilization of HGA-MT will facilitate efforts to purify the
enzyme, to study the structure of HGA-MT, and to clone the gene. The
HGA-MT gene should facilitate the study of the proposed pectin-biosynthetic complex and allow the manipulation of HGA-MT genes
in transgenic plants to study the role of HGA methylation in plant
growth and development.
| |
FOOTNOTES |
1
This work was supported by a grant from the
Hercules Corporation, Wilmington, DE.
2
Present address: Institut National de la
Recherche Agronomique, Laboratoire de Biologie Cellulaire, Route
de Saint Cyr, 78026 Versailles cedex, France.
*
Corresponding author; e-mail dmohnen{at}ccrc.uga.edu; fax
706-542-4412.
Received February 9, 1999;
accepted June 4, 1999.
| |
ACKNOWLEDGMENTS |
We thank the Hercules Corporation for funding this work. We also
thank Carl Bergmann for many helpful suggestions and for the gifts of
purified EPGase from A. niger, purified EPGase from F. moniliforme, and the purified PME. We thank Hans Peter
Heldt-Hansen for the gift of cloned PME. Elizabeth Dabbs Loomis is
gratefully acknowledged for the size-exclusion chromatography of
pectin. We thank Stefan Eberhard for the gift of tobacco cell
suspensions, Carol L. Gubbins-Hahn for drawing the figures, Malcolm
O'Neill for critical reading of the manuscript, and our colleagues at the Complex Carbohydrate Research Center for their helpful discussions.
| |
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Abstract
Introduction
